Project Summary: Intestinal homeostasis is crucial for normal intestinal functions, and its disruption may lead to the pathogenesis of human diseases such as chronic inflammation and even cancer. Paneth cells play crucial roles in intestinal barrier function and mucosal immunity. Recent studies have demonstrated that impaired function of Paneth cells may lead to dysbiotic intestine and increased susceptibility to inflammatory bowel disease (IBD). The Ufm1 (Ubiquitin-fold modifier 1) conjugation system is a novel ubiquitin-like system that consists of Ufm1- sepcific E1 enzyme (Uba5), E2 enzyme (Ufc1) and a putative E3 enzyme (a protein complex containing Ufl1 and Ufbp1). Clinical and animal model studies implicate its potential involvement in many diseases such as neurological disorders, ischemic injury and cancer, but its overall functions and underlying mechanisms remain poorly understood. Genetic studies from others and us have recently demonstrated the essential role of this system in embryonic development and hematopoiesis, but its role in other systems remains to be defined. Ufl1 and Ufbp1, the key components of Ufm1 E3 ligase, are present in multiple tissues and organs and highly expressed in the intestine, yet their role in the gut is completely unknown. The observation of frequent gut bleeding in Ufl1 and Ufbp1 knockout mice prompted us to generate IEC (intestinal epithelial cell)-specific knockout mouse models of Ufl1 and Ufbp1. We found that IEC-specific ablation of either Ufl1 or Ufbp1 caused nearly complete loss of Paneth cells and partial loss of goblet cells, resulting in increased susceptibility to experimental colitis. Moreover, Ufbp1 ablation led to elevated ER (endoplasmic reticulum) stress, activation of the Unfolded Protein Response (UPR) and apoptosis in IECs. We also observed dramatic increase of mRNAs of secretory proteins such as lysozyme during the course of acute Ufbp1 ablation. We hypothesize that Ufbp1 and the ufmylation pathway plays a pivotal role in IRE1-mediated degradation of ER-associated mRNAs, thereby coordinating protein synthesis and ER protein load and maintaining ER homeostasis of Paneth cells. Ufbp1 deficiency impairs this degradation mechanism and leads to increased mRNAs and protein translation, resulting in elevated ER stress and Paneth cell death. To test this hypothesis, we propose three specific aims. Aim 1: To define Paneth cell-specific function of Ufbp1; Aim 2: To investigate the role of UPR in Paneth cell apoptosis induced by Ufbp1 deficiency; and Aim 3: To elucidate the mechanism of Ufbp1 ablation-induced disruption of ER homeostasis. The outcome of this project will provide critical insight into the physiological function and working mechanism of this important protein modification system in maintaining intestinal homeostasis., and have a significant impact on our understanding of Paneth cell biology, development of novel therapeutic targets for inflammatory disease, and the physiological function of the ufmylation pathway and its interplay with UPR.